Glycerol-3-phosphate acyltransferase 6 controls filamentous pathogen interactions and cell wall properties of the tomato and Nicotiana benthamiana leaf epidermis.

The leaf outer epidermal cell wall acts as a barrier against pathogen attack and desiccation, and as such is covered by a cuticle, composed of waxes and the polymer cutin. Cutin monomers are formed by the transfer of fatty acids to glycerol by glycerol-3-phosphate acyltransferases, which facilitate their transport to the surface. The extent to which cutin monomers affect leaf cell wall architecture and barrier properties is not known. We report a dual functionality of pathogen-inducible GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE 6 (GPAT6) in controlling pathogen entry and cell wall properties affecting dehydration in leaves. Silencing of Nicotiana benthamiana NbGPAT6a increased leaf susceptibility to infection by the oomycetes Phytophthora infestans and Phytophthora palmivora, whereas overexpression of NbGPAT6a-GFP rendered leaves more resistant. A loss-of-function mutation in tomato SlGPAT6 similarly resulted in increased susceptibility of leaves to Phytophthora infection, concomitant with changes in haustoria morphology. Modulation of GPAT6 expression altered the outer wall diameter of leaf epidermal cells. Moreover, we observed that tomato gpat6-a mutants had an impaired cell wall-cuticle continuum and fewer stomata, but showed increased water loss. This study highlights a hitherto unknown role for GPAT6-generated cutin monomers in influencing epidermal cell properties that are integral to leaf-microbe interactions and in limiting dehydration.Royal Society (RG120398, UF110073, UF160413) and the Gatsby Charitable Foundation (GAT3395/GLD) Plant Genome Research Program of the US National Science Foundation (IOS-1339287) Agriculture and Food Research Initiative of the US Department of Agriculture (2016-67013-24732)

Alginates along the filament of the brown alga Ectocarpus help cells cope with stress.

Ectocarpus is a filamentous brown alga, which cell wall is composed mainly of alginates and fucans (80%), two non-crystalline polysaccharide classes. Alginates are linear chains of epimers of 1,4-linked uronic acids, β-D-mannuronic acid (M) and α-L-guluronic acid (G). Previous physico-chemical studies showed that G-rich alginate gels are stiffer than M-rich alginate gels when prepared in vitro with calcium. In order to assess the possible role of alginates in Ectocarpus, we first immunolocalised M-rich or G-rich alginates using specific monoclonal antibodies along the filament. As a second step, we calculated the tensile stress experienced by the cell wall along the filament, and varied it with hypertonic or hypotonic solutions. As a third step, we measured the stiffness of the cell along the filament, using cell deformation measurements and atomic force microscopy. Overlapping of the three sets of data allowed to show that alginates co-localise with the stiffest and most stressed areas of the filament, namely the dome of the apical cell and the shanks of the central round cells. In addition, no major distinction between M-rich and G-rich alginate spatial patterns could be observed. Altogether, these results support that both M-rich and G-rich alginates play similar roles in stiffening the cell wall where the tensile stress is high and exposes cells to bursting, and that these roles are independent from cell growth and differentiation

Monoclonal antibodies indicate low-abundance links between heteroxylan and other glycans of plant cell walls.

The derivation of two sensitive monoclonal antibodies directed to heteroxylan cell wall polysaccharide preparations has allowed the identification of potential inter-linkages between xylan and pectin in potato tuber cell walls and also between xylan and arabinogalactan-proteins in oat grain cell walls. Plant cell walls are complex composites of structurally distinct glycans that are poorly understood in terms of both in muro inter-linkages and developmental functions. Monoclonal antibodies (MAbs) are versatile tools that can detect cell wall glycans with high sensitivity through the specific recognition of oligosaccharide structures. The isolation of two novel MAbs, LM27 and LM28, directed to heteroxylan, subsequent to immunisation with a potato cell wall fraction enriched in rhamnogalacturonan-I (RG-I) oligosaccharides, is described. LM27 binds strongly to heteroxylan preparations from grass cell walls and LM28 binds to a glucuronosyl-containing epitope widely present in heteroxylans. Evidence is presented suggesting that in potato tuber cell walls, some glucuronoxylan may be linked to pectic macromolecules. Evidence is also presented that suggests in oat spelt xylan both the LM27 and LM28 epitopes are linked to arabinogalactan-proteins as tracked by the LM2 arabinogalactan-protein epitope. This work extends knowledge of the potential occurrence of inter-glycan links within plant cell walls and describes molecular tools for the further analysis of such links.This work was supported by the European Union Seventh Framework Programme (FP7 2007-2013) under the WallTraC project (Grant Agreement number 263916). (This article reflects the authors’ views only and the European Union is not liable for any use that may be made of the information contained herein). The work was also supported by the United Kingdom Biotechnology and Biological Research Council (BBSRC, Grant BB/K017489/1). JX acknowledges support from the Chinese Scholarship Council, TAT from a BBSRC studentship and MGR from the Danish Strategic Research Council and The Danish Council for Independent Research, Technology and Production Sciences as part of the GlycAct project (FI 10-093465). We acknowledge kind gifts of enzymes from Harry Gilbert and oligosaccharides from Sanna Koutaniemi. We thank Theodora Tryfona for mass spectrometry analysis.This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s00425-015-2375-

Dissection of cell wall assembly dynamics during early embryogenesis in the brown alga Fucus

Zygotes from Fucus species have been used extensively to study cell polarization and rhizoid outgrowth, and in this model system cell wall deposition aligns with the establishment of polarity. Monoclonal antibodies are essential tools for the in situ analysis of cell wall glycans, and here we report the characteristics of six monoclonal antibodies to alginates (BAM6–BAM11). The use of these, in conjunction with monoclonal antibodies to brown algal sulfated fucans, has enabled the study of the developmental dynamics of the Fucus zygote cell walls. Young zygotes are spherical and all alginate epitopes are deposited uniformly following cellulose deposition. At germination, sulfated fucans are secreted in the growing rhizoid wall. The redistribution of cell wall epitopes was investigated during treatments that cause reorientation of the growth axis (change in light direction) or disrupt rhizoid development (arabinogalactan-protein-reactive Yariv reagent). Alginate modeling was drastically impaired in the latter, and both treatments cause a redistribution of highly sulfated fucan epitopes. The dynamics of cell wall glycans in this system have been visualized in situ for the first time, leading to an enhanced understanding of the early developmental mechanisms of Fucus species. These sets of monoclonal antibodies significantly extend the available molecular tools for brown algal cell wall studies

Monoclonal antibodies directed to fucoidan preparations from brown algae

Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance

Epitope detection chromatographic (EDC) anion-exchange analysis.

<p>EDC analysis of A, sulfated (DS0) and B, de-sulfated (DS3) derivatives of FS28 using BAM1, BAM2, BAM4 and LM23 as detection tools. The elution gradient was 0 to 4 M NaCl from 26 ml to 80 ml elution volume. Blue arrows indicate shift in peak of BAM1 epitope elution associated with de-sulfation. Green arrows indicate loss of BAM4 epitope peak height but no shift in elution time. EDC profiles shown are representative of two chromatographic runs.</p

Analysis of <i>Ectocarpus subulatus</i>.

<p>Analysis of <i>E</i>. <i>subulatus</i> grown in seawater (100%) or in 20-fold diluted seawater (5%). (<b>A</b>) Indirect immunofluorescence detection of the BAM4 epitope at the surface of whole mount preparations, scale = 25 μm. (<b>B</b>) ELISA analysis of BAM4 epitope levels in extracts of AIR. C = CaCl₂ extract, N = Na₂CO₃ extract, K = KOH extract. (<b>C</b>) Sulfate levels (FS28 equivalents w/w) in C, N and K extracts of AIR. Error bars indicate SD of 4 replicates.</p

Heat map of antibody binding to cell wall extracts of a range of brown algae.

<p>Heat map of relative BAM1 to BAM4, LM7 and LM23 epitope levels as determined by ELISA absorbance obtained from 25-fold dilution of MAbs with 50 μg/ml of extracts from 8 species of the Fucales (F), 6 species of Laminariales (L) and 1 species of Ectocarpales (E). Fucale and Laminariale species are listed in relation to approximate occurrence from upper to lower regions of intertidal zones. Extracts of AIR with CaCl₂ (C), Na₂CO₃ (N) and KOH (K) after dialysis and freeze-drying. The colour scale in relation to absorbance values is shown top left. Values shown are means of 4 replicates and in all cases SDs were <0.1 absorbance units.</p

Heat map showing the relative binding of the BAM1 to BAM4, LM7 and LM23 MAbs to a range of brown algal, sulfated, red algal and land plant polysaccharides.

<p>Binding was determined by ELISA with 50 μg/ml of polysaccharide samples and 25-fold dilutions of antibody hybridoma supernatants. S indicates polysaccharides containing sulfate residues. The colour scale in relation to absorbance values is shown top left. Values shown are means of 4 replicates and in all cases standard deviations (SDs) were <0.1 absorbance units.</p